CN105331726A - Method for sequencing human mitochondria genetic set based on sanger - Google Patents
Method for sequencing human mitochondria genetic set based on sanger Download PDFInfo
- Publication number
- CN105331726A CN105331726A CN201510863948.4A CN201510863948A CN105331726A CN 105331726 A CN105331726 A CN 105331726A CN 201510863948 A CN201510863948 A CN 201510863948A CN 105331726 A CN105331726 A CN 105331726A
- Authority
- CN
- China
- Prior art keywords
- seqidno
- primer pair
- human
- sanger
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000003470 mitochondria Anatomy 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 25
- 230000002068 genetic effect Effects 0.000 title abstract description 7
- 238000012163 sequencing technique Methods 0.000 title abstract 3
- 108020004414 DNA Proteins 0.000 claims abstract description 16
- 108020005196 Mitochondrial DNA Proteins 0.000 claims abstract description 15
- 238000012408 PCR amplification Methods 0.000 claims abstract description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 51
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 239000012634 fragment Substances 0.000 claims description 8
- 238000011084 recovery Methods 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000012772 sequence design Methods 0.000 claims description 3
- 238000007400 DNA extraction Methods 0.000 claims description 2
- 238000001712 DNA sequencing Methods 0.000 abstract 1
- 239000000706 filtrate Substances 0.000 description 6
- 239000000284 extract Substances 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 208000012268 mitochondrial disease Diseases 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 101150001086 COB gene Proteins 0.000 description 1
- 102100028203 Cytochrome c oxidase subunit 3 Human genes 0.000 description 1
- 108010075028 Cytochromes b Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 206010011878 Deafness Diseases 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101000861034 Homo sapiens Cytochrome c oxidase subunit 3 Proteins 0.000 description 1
- 101001109060 Homo sapiens NADH-ubiquinone oxidoreductase chain 4L Proteins 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 101150053771 MT-CYB gene Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010052641 Mitochondrial DNA mutation Diseases 0.000 description 1
- 206010058799 Mitochondrial encephalomyopathy Diseases 0.000 description 1
- 102100021452 NADH-ubiquinone oxidoreductase chain 4L Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 101710157860 Oxydoreductase Proteins 0.000 description 1
- 208000006735 Periostitis Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000712 assembly Effects 0.000 description 1
- 238000000429 assembly Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 101150006264 ctb-1 gene Proteins 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 231100000895 deafness Toxicity 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 208000016354 hearing loss disease Diseases 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 208000014188 hereditary optic neuropathy Diseases 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 101150087530 mt:ATPase6 gene Proteins 0.000 description 1
- 101150078624 mt:ATPase8 gene Proteins 0.000 description 1
- 101150088166 mt:Cyt-b gene Proteins 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 238000001668 nucleic acid synthesis Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000002220 organoid Anatomy 0.000 description 1
- 210000003460 periosteum Anatomy 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 230000011218 segmentation Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The invention discloses a method for sequencing a human mitochondria genetic set based on sanger. The method comprises the steps that total human DNA is extracted; primer pairs covering the overall length of the mitochondria are designed on the basis of the human mitochondria sequence measured in an NCBI database, and PCR amplification is performed on the total human DNA; a PCR amplification product is recycled to obtain genetic segments specified by the human mitochondria; the genetic segments are sequenced; CExpress is used for splicing the measured sequences according to sequence overlaid areas between the primer pairs, and assembling is performed to obtain the total human mitochondria sequence. According to the method, only the total human DNA needs to be extracted, the complex experimental operation process that the high-purity mtDNA is extracted is avoided, then the primer pairs covering the total length of mitochondria are designed according to the human mitochondria sequence measured in the NCBI database for amplifying the total DNA to obtain the specific genetic segments of the mitochondria for sequencing, and the data amount of the total DNA sequencing is reduced.
Description
Technical field
The present invention relates to genetically engineered field, be specifically related to a kind of method of the human mitochondria gene group that checks order based on sanger.
Background technology
Since plastosome self-discovery, its morphological structure, genomic constitution, copies, and transcribes and translation, with the relation of Matrix attachment region, and its genetic characteristics, evolution feature and molecular systematics aspect all have accumulated a large amount of data.Plastosome is organoid important in eukaryotic cell, the place that energy generates, and also participates in the synthesis of lipid acid and the synthesis of some protein.Mitochondrial DNA is genome relatively independent in cell.Mitochondrial DNA be less in cell and easier purifying copy transcription unit, genome structure is fairly simple, and there is very high specificity, and unique, its transmission, restructuring, be separated, copy, transcribe and all can many measures of applied molecular biology analyze.Therefore, the good model that Mitochondrial DNA is not only researching DNA structure and DNA replication dna, is transcribed also is the non-suitable model system of general considerations such as research eukaryotic cell nucleic acid and protein synthesis etc.Measure the complete sequence of human mitochondria gene group from (1981) such as Anderson since, human mitochondrion is a covalence closed double-stranded DNA, molecular weight, and length is 16569 bases.Human mitochondria gene group is altogether containing 37 genes.22 tRNA genes, cytochrome b gene (Cytb), Terminal oxidase 3 subunit genes (COI, COII, COIII), NADH oxydo-reductase 7 subunit genes (ND1, ND2, ND3, ND4, ND4L, ND5, and ATP enzyme 2 subunit gene (ATPase6 ND6), ATPase8), a 12SrRNA gene, a 16SrRNA gene.
The sudden change of Mitochondrial DNA is relevant to many human diseasess, and the class disease caused by Mitochondrial DNA Mutation is called as mitochondrial disease.The generation of disease, development and genetic development thereof can be disclosed from molecular biological angle research mitochondrial disease, thus provide scientific basis for the diagnosis of disease, treatment and prevention.The mitochondrial disease that current research finds mainly contains Lerber ' s hereditary optic neuropathy, mitochondrial encephalomyopathy, the diabetes of matrilinear inheritance and deafness etc.
Existing multiple line mitochondrial genes sequence measurement, as the sanger sequencing based on clone library, PCR-based increases in advance or extracts the high-flux sequence method of mtDNA, high-flux sequence method etc. based on STb gene, these sequence measurement keys are the mtDNA being to obtain higher degree, or need the original data volume of order-checking higher.But obtain the mtDNA of higher degree, operate more loaded down with trivial details, experimentation is careless slightly, and the mtDNA obtained has core DNA pollution, and a large amount of original data volume can increase considerably the workload of order-checking.
Summary of the invention
The object of the invention is to the deficiency overcoming the existence of above-mentioned prior art, a kind of method of the human mitochondria gene group that checks order based on sanger is provided.
The object of the invention is to be achieved through the following technical solutions:
The present invention relates to a kind of method of the human mitochondria gene group that checks order based on sanger, described method comprises the steps:
S1, people's total DNA extraction;
S2, cover the primer pair of plastosome total length according to the human mitochondrion sequence design of having measured in ncbi database, pcr amplification is carried out to people's STb gene;
S3, recovery pcr amplification product, obtain the specific gene fragment of human mitochondrion;
S4, described gene fragment to be checked order; Splice according to the overlapping sequences district between primer pair and primer pair the sequence recorded with CExpress, assembling obtains human mitochondrion complete sequence.
Preferably, in step S2, described primer pair comprises the primer pair that the HS-12S-F as shown in SEQIDNO.1 and the HS-12S-R as shown in SEQIDNO.2 is formed, the primer pair that HS-16S-F as shown in SEQIDNO.3 and the HS-16S-R as shown in SEQIDNO.4 is formed, the primer pair that HS-C1-F as shown in SEQIDNO.5 and the HS-C1-R as shown in SEQIDNO.6 is formed, the primer pair that HS-C2-F as shown in SEQIDNO.7 and the HS-C2-R as shown in SEQIDNO.8 is formed, the primer pair that HS-N2-F as shown in SEQIDNO.9 and the HS-N2-R as shown in SEQIDNO.10 is formed, the primer pair that HS-CB-F as shown in SEQIDNO.11 and the HS-CB-R as shown in SEQIDNO.12 is formed, the primer pair that HS-C3-F as shown in SEQIDNO.13 and the HS-C3-R as shown in SEQIDNO.14 is formed, the primer pair that HS-N4-F as shown in SEQIDNO.15 and the HS-N4-R as shown in SEQIDNO.16 is formed, the primer pair that HS-N5-F as shown in SEQIDNO.17 and the HS-N5-R as shown in SEQIDNO.28 is formed, the primer pair that HS-12S-S as shown in SEQIDNO.19 and the HS-16S-C as shown in SEQIDNO.20 is formed, the primer pair that HS-N2-S as shown in SEQIDNO.21 and the HS-C1-C as shown in SEQIDNO.22 is formed, the primer pair that HS-N1-F as shown in SEQIDNO.23 and the HS-N1-R as shown in SEQIDNO.24 is formed, the primer pair that HS-CB-S as shown in SEQIDNO.25 and the HS-12S-C as shown in SEQIDNO.26 is formed, the primer pair that HS-C2-S as shown in SEQIDNO.27 and the HS-C3-C as shown in SEQIDNO.28 is formed, the primer pair that HS-C3-S as shown in SEQIDNO.29 and the HS-N4-C as shown in SEQIDNO.30 is formed, the primer pair that HS-N4-S as shown in SEQIDNO.31 and the HS-N5-C as shown in SEQIDNO.32 is formed, the primer pair that HS-N5-S as shown in SEQIDNO.33 and the HS-CB-C as shown in SEQIDNO.34 is formed, the primer pair that HS-C1-S as shown in SEQIDNO.35 and the HS-C2-C as shown in SEQIDNO.36 is formed, the primer pair that HS-16S-S as shown in SEQIDNO.37 and the HS-N1-C as shown in SEQIDNO.38 is formed.
Preferably, in step S2, in described pcr amplification, every 50.0ul amplification reaction system comprises: genomic dna 1.0ul, containing the LATaq polysaccharase 1.0ul of the 10 × LABuffer5.0ul of 2.5mMMg2+, 5u/ μ L, the dNTP1.0ul of 10mM, the reverse primer 1.5ul of the forward primer 1.5ul of 10uM, 10uM, ddH2O39.0ul.
Preferably, in step S2, in described pcr amplification, PCR reaction parameter is: denaturation 95 DEG C, 5min; Sex change 95 DEG C, 30s; Anneal 55 DEG C, 45s; Extend 72 DEG C, 1min30s; Extend 72 DEG C eventually, 7m; Cycle number 45.
Preferably, in step S3, described recovery reclaims test kit with AxyPrepDNA gel to reclaim.
Preferably, in step S4, described order-checking carries out generation order-checking based on Sanger dideoxy chain termination.
Preferably, step S4 also comprise for assemble the human mitochondrion complete sequence that obtains according to the determination of the human mitochondrion sequence direction of travel measured and zero point gene the step of determination.
Preferably, being defined as of described direction: determine that whether gene direction is consistent with other human mitochondrion sequence, if unanimously, then do not need adjustment direction, otherwise, need sequence reverse complemental.
Preferably, being defined as of described gene at zero point: on the basis determining mtDNA sequence direction, using the zero position of first gene as first base, other sequences arrange in order.
The ultimate principle of method of the present invention designs by known person mtDNA sequence the primer pair covering plastosome total length, the specific gene fragment of segmentation amplification plastosome, then measure the sequence of these genes with ABI3730xl sequenator, then these sequence assemblies are become a complete plastosome by the overlap between sequence with sequence.
Compared with prior art, the present invention has following beneficial effect:
The present invention only need extract people's STb gene, avoid and extract the loaded down with trivial details experimental implementation process of higher degree mtDNA, then the primer pair of plastosome total length is covered according to the human mitochondrion sequences Design measured in ncbi database, STb gene is increased, obtain the specific gene fragment of plastosome, check order, decrease the data volume for STb gene order-checking.
Accompanying drawing explanation
Fig. 1 is the effect schematic diagram that the sequence recorded carries out according to the overlapping sequences district between primer pair and primer pair splicing.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make certain adjustments and improvements.These all belong to protection scope of the present invention.
embodiment
The present embodiment relates to a kind of method of the human mitochondria gene group that checks order based on sanger; Specifically comprise the steps:
One, genome extracts: extract people's STb gene
1. get 1-20mg animal tissues, move in the mortar of ice-water bath precooling, grind to form homogenate fast, firmly.
2. after adding 350 μ lBufferPBS and 0.9 μ lRNaseA, leniently grind 30s.
3. collect the ground tissue homogenate of 350 μ l and proceed to 2ml centrifuge tube.If homogenate volume is less than 350 μ l, supplement PBS to 350 μ l.
4. add 150 μ lBufferC-L and 20 μ lProteinaseK.Vortex oscillation 1min mixes immediately.Of short duration centrifugal after, centrifuge tube is put 56 DEG C of water-bath 10min.
* ProteinaseK is not directly added in BufferC-L.
5. add 350 μ lBufferP-D, vortex oscillation 30s mixes, 12,000 × g centrifugal 10min.
6. prepared by DNA pipe and be placed in 2ml centrifuge tube, the mixed solution in step 5 is moved to and prepares in pipe, 12,000 × g centrifugal 1min.
7. abandon filtrate, put get back to preparing pipe in original 2ml centrifuge tube, add 500 μ lBufferW1,12,000 × g centrifugal 1min.
8. abandon filtrate, put get back to preparing pipe in original 2ml centrifuge tube, add 700 μ lBufferW2,12,000 × g centrifugal 1min, in the same way, wash again once with 700 μ lBufferW2.
* confirm to add dehydrated alcohol by the volume that reagent bottle is specified in BufferW2concentrate.
* again rinse with BufferW2 and can guarantee that salt is completely removed, eliminate the impact on endonuclease reaction.
9. abandoning filtrate, putting back in original 2ml centrifuge tube by preparing pipe, 12,000 × g centrifugal 1min.
10. prepared by DNA the 1.5ml centrifuge tube that pipe is placed in another cleaning, add 100-200 μ lEluent or deionized water preparing periosteum central authorities, room temperature leaves standstill 1min, 12,000 × g centrifugal 1min eluted dnas.
* deionized water or Eluent are heated to 65 DEG C and will improve elution efficiency.
Two, design of primers and synthesis
Cover the primer pair of plastosome total length according to the human mitochondrion sequence design of having measured in ncbi database, these primers are synthesized by upper Shanghai's style Sen Nuo bio tech ltd, and primer sequence is as follows:
Three, pcr amplification: increase to STb gene, obtains the specific gene fragment of plastosome
3.1PCR amplification reaction system
Table 1
| Genomic dna | 1.0ul |
| 10 × LA Buffer (containing 2.5mM Mg2+) | 5.0ul |
| LA Taq polysaccharase (5u/ μ L) | 1.0ul |
| dNTP(10mM) | 1.0ul |
| Forward primer (10uM) | 1.5ul |
| Reverse primer (10uM) | 1.5ul |
| ddH2O | 39.0ul |
| Cumulative volume | 50.0ul |
In 0.2ml centrifuge tube, add composition shown in table 1, mixing, the drop on brief centrifugation collection tube wall is at the bottom of pipe, and in the enterprising performing PCR reaction of PCR amplification instrument, reaction parameter is as shown in table 2 below:
Table 2
| Denaturation | Sex change | Annealing | Extend | Extend eventually | Cycle number |
| 95℃，5min | 95℃，30s | 65℃，30s | 72℃，1min30s | 72℃，7m | 45 |
After having reacted, get 3ulPCR product and carry out 1% agarose gel electrophoresis detection.Confirm pcr amplified fragment.
The recovery of 3.2PCR product
PCR primer reclaims test kit with AxyPrepDNA gel and reclaims, and concrete operations are undertaken by test kit specification sheets, and step is as follows:
3.2.1 under ultraviolet lamp, clean centrifuge tube put into by the sepharose cut containing target DNA, takes weight.
3.2.2 the BufferDE-A of 3 gel volumes is added, in 75 DEG C of heating until gel piece melts completely after mixing.
3.2.3 add the BufferDE-B of 0.5 BufferDE-A volume, mix; When the DNA fragmentation be separated is less than 400bp, add the Virahol of 1 gel volume.
3.2.4 by mixed solution, transfer to DNA and prepare pipe 12, the centrifugal 1min of 000 × g.Abandon filtrate.
3.2.5 putting back 2ml centrifuge tube by preparing pipe, adding 500 μ lBufferW1,12,000 × g centrifugal 30s, abandon filtrate.
3.2.6 putting back 2ml centrifuge tube by preparing pipe, adding 700 μ lBufferW2,12,000 × g centrifugal 30s, abandon filtrate.Use 700 μ lBufferW2 in the same way again, 12,000 × g centrifugal 1min.
3.2.7 put back in 2ml centrifuge tube by preparing pipe, 12,000 × g centrifugal 1min.
3.2.8 be placed in clean 1.5ml centrifuge tube (providing in test kit) by preparing pipe, add 25-30 μ l deionized water preparing film central authorities, room temperature leaves standstill 1min.12,000 × g centrifugal 1min eluted dnas.
Four, generation order-checking
Utilize and carry out generation order-checking based on the technology of Sanger dideoxy chain termination, sequenator used is ABI3730xl.
Five, sequence assembly
With CExpress, the sequence recorded is spliced according to the overlapping sequences district between primer pair and primer pair, splicing effect as shown in Figure 1, as shown in Figure 1, the sequence recorded is spliced according to the overlapping sequences district between primer pair and primer pair, obtain complete plastosome complete sequence.
But for closed hoop molecule, linear molecule can be fragmented in any one place, in addition, software combination be also do not have directive.Therefore for assembling the human mitochondrion complete sequence that obtains, need according to the determination of the human mitochondrion sequence direction of travel measured and zero point gene determination.
The determination in direction: determine that whether gene direction is consistent with other human mitochondrion sequence, if unanimously, then do not need adjustment direction, otherwise, need sequence reverse complemental.
Zero point gene determination: on the basis determining mtDNA sequence direction, using the zero position of first gene as first base, other sequences arrange in order.
Claims (9)
1. a method for the human mitochondria gene group that checks order based on sanger, it is characterized in that, described method comprises the steps:
S1, people's total DNA extraction;
S2, cover the primer pair of plastosome total length according to the human mitochondrion sequence design of having measured in ncbi database, pcr amplification is carried out to people's STb gene;
S3, recovery pcr amplification product, obtain the specific gene fragment of human mitochondrion;
S4, described gene fragment to be checked order; Splice according to the overlapping sequences district between primer pair and primer pair the sequence recorded with CExpress, assembling obtains human mitochondrion complete sequence.
2. the method for the human mitochondria gene group that checks order based on sanger according to claim 1, it is characterized in that, in step S2, described primer pair comprises the primer pair that the HS-12S-F as shown in SEQIDNO.1 and the HS-12S-R as shown in SEQIDNO.2 is formed, the primer pair that HS-16S-F as shown in SEQIDNO.3 and the HS-16S-R as shown in SEQIDNO.4 is formed, the primer pair that HS-C1-F as shown in SEQIDNO.5 and the HS-C1-R as shown in SEQIDNO.6 is formed, the primer pair that HS-C2-F as shown in SEQIDNO.7 and the HS-C2-R as shown in SEQIDNO.8 is formed, the primer pair that HS-N2-F as shown in SEQIDNO.9 and the HS-N2-R as shown in SEQIDNO.10 is formed, the primer pair that HS-CB-F as shown in SEQIDNO.11 and the HS-CB-R as shown in SEQIDNO.12 is formed, the primer pair that HS-C3-F as shown in SEQIDNO.13 and the HS-C3-R as shown in SEQIDNO.14 is formed, the primer pair that HS-N4-F as shown in SEQIDNO.15 and the HS-N4-R as shown in SEQIDNO.16 is formed, the primer pair that HS-N5-F as shown in SEQIDNO.17 and the HS-N5-R as shown in SEQIDNO.28 is formed, the primer pair that HS-12S-S as shown in SEQIDNO.19 and the HS-16S-C as shown in SEQIDNO.20 is formed, the primer pair that HS-N2-S as shown in SEQIDNO.21 and the HS-C1-C as shown in SEQIDNO.22 is formed, the primer pair that HS-N1-F as shown in SEQIDNO.23 and the HS-N1-R as shown in SEQIDNO.24 is formed, the primer pair that HS-CB-S as shown in SEQIDNO.25 and the HS-12S-C as shown in SEQIDNO.26 is formed, the primer pair that HS-C2-S as shown in SEQIDNO.27 and the HS-C3-C as shown in SEQIDNO.28 is formed, the primer pair that HS-C3-S as shown in SEQIDNO.29 and the HS-N4-C as shown in SEQIDNO.30 is formed, the primer pair that HS-N4-S as shown in SEQIDNO.31 and the HS-N5-C as shown in SEQIDNO.32 is formed, the primer pair that HS-N5-S as shown in SEQIDNO.33 and the HS-CB-C as shown in SEQIDNO.34 is formed, the primer pair that HS-C1-S as shown in SEQIDNO.35 and the HS-C2-C as shown in SEQIDNO.36 is formed, the primer pair that HS-16S-S as shown in SEQIDNO.37 and the HS-N1-C as shown in SEQIDNO.38 is formed.
3. the method for the human mitochondria gene group that checks order based on sanger according to claim 1, it is characterized in that, in step S2, in described pcr amplification, every 50.0ul amplification reaction system comprises: genomic dna 1.0ul, containing the 10 × LABuffer5.0ul of 2.5mMMg2+, the LATaq polysaccharase 1.0ul of 5u/ μ L, the forward primer 1.5ul of the dNTP1.0ul of 10mM, 10uM, the reverse primer 1.5ul of 10uM, ddH2O39.0ul.
4. the method for the human mitochondria gene group that checks order based on sanger according to claim 1, it is characterized in that, in step S2, in described pcr amplification, PCR reaction parameter is: denaturation 95 DEG C, 5min; Sex change 95 DEG C, 30s; Anneal 55 DEG C, 45s; Extend 72.C, 1min30s; Extend 72 DEG C eventually, 7m; Cycle number 45.
5. the method for the human mitochondria gene group that checks order based on sanger according to claim 1, is characterized in that, in step S3, described recovery reclaims test kit with AxyPrepDNA gel to reclaim.
6. the method for the human mitochondria gene group that checks order based on sanger according to claim 1, it is characterized in that, in step S4, described order-checking carries out generation order-checking based on Sanger dideoxy chain termination.
7. the method for the human mitochondria gene group that checks order based on sanger according to claim 1, it is characterized in that, step S4 also comprise for assemble the human mitochondrion complete sequence that obtains according to the determination of the human mitochondrion sequence direction of travel measured and zero point gene the step of determination.
8. the method for the human mitochondria gene group that checks order based on sanger according to claim 7, is characterized in that, being defined as of described direction: determine that whether gene direction is consistent with other human mitochondrion sequence, if consistent, then do not need adjustment direction, otherwise, need sequence reverse complemental.
9. the method for the human mitochondria gene group that checks order based on sanger according to claim 7, it is characterized in that, being defined as of described gene at zero point: on the basis determining mtDNA sequence direction, using the zero position of first gene as first base, other sequences arrange in order.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510863948.4A CN105331726B (en) | 2015-12-01 | 2015-12-01 | Method based on sanger sequencing human mitochondria gene group |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510863948.4A CN105331726B (en) | 2015-12-01 | 2015-12-01 | Method based on sanger sequencing human mitochondria gene group |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105331726A true CN105331726A (en) | 2016-02-17 |
| CN105331726B CN105331726B (en) | 2019-02-12 |
Family
ID=55282489
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510863948.4A Active CN105331726B (en) | 2015-12-01 | 2015-12-01 | Method based on sanger sequencing human mitochondria gene group |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105331726B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636359A (en) * | 2016-11-15 | 2017-05-10 | 上海派森诺生物科技股份有限公司 | Sanger-based method for sequencing recombinant HPV (human papillomavirus) adenovirus genome |
| CN106755456A (en) * | 2017-01-09 | 2017-05-31 | 北京圣谷智汇医学检验所有限公司 | For the primer combination of mitochondria full-length genome detection and kit |
| CN108315399A (en) * | 2018-01-19 | 2018-07-24 | 成都新基因格生物科技有限公司 | Chondriogen detection kit and application method |
| CN111621552A (en) * | 2019-06-13 | 2020-09-04 | 中国科学院广州生物医药与健康研究院 | Method and system for detecting mtDNA mutation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270390A (en) * | 2008-04-15 | 2008-09-24 | 西安交通大学 | 26-pair PCR primer for mitochondrion sequencing and parting method based on the primer |
| CN101875966A (en) * | 2009-04-30 | 2010-11-03 | 海南大学 | Improved mitochondrial genome complete sequence determination method |
| CN104480207A (en) * | 2014-12-17 | 2015-04-01 | 杭州吉洛生物医药科技有限公司 | Primer pair capable of detecting specificity of human mitochondrial genome |
-
2015
- 2015-12-01 CN CN201510863948.4A patent/CN105331726B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270390A (en) * | 2008-04-15 | 2008-09-24 | 西安交通大学 | 26-pair PCR primer for mitochondrion sequencing and parting method based on the primer |
| CN101875966A (en) * | 2009-04-30 | 2010-11-03 | 海南大学 | Improved mitochondrial genome complete sequence determination method |
| CN104480207A (en) * | 2014-12-17 | 2015-04-01 | 杭州吉洛生物医药科技有限公司 | Primer pair capable of detecting specificity of human mitochondrial genome |
Non-Patent Citations (1)
| Title |
|---|
| 沙淼等: "线粒体基因组测序策略和方法", 《应用昆虫学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636359A (en) * | 2016-11-15 | 2017-05-10 | 上海派森诺生物科技股份有限公司 | Sanger-based method for sequencing recombinant HPV (human papillomavirus) adenovirus genome |
| CN106636359B (en) * | 2016-11-15 | 2020-08-18 | 上海派森诺生物科技股份有限公司 | Method for sequencing recombinant HPV adenovirus genome based on sanger sequencing |
| CN106755456A (en) * | 2017-01-09 | 2017-05-31 | 北京圣谷智汇医学检验所有限公司 | For the primer combination of mitochondria full-length genome detection and kit |
| CN106755456B (en) * | 2017-01-09 | 2020-02-21 | 北京圣谷智汇医学检验所有限公司 | Primer combination and kit for mitochondrial whole genome detection |
| CN108315399A (en) * | 2018-01-19 | 2018-07-24 | 成都新基因格生物科技有限公司 | Chondriogen detection kit and application method |
| CN111621552A (en) * | 2019-06-13 | 2020-09-04 | 中国科学院广州生物医药与健康研究院 | Method and system for detecting mtDNA mutation |
| CN111621552B (en) * | 2019-06-13 | 2022-05-06 | 中国科学院广州生物医药与健康研究院 | Method and system for detecting mtDNA mutation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105331726B (en) | 2019-02-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103180459B (en) | The sequencing strategy in 3-D genes of interest group region | |
| Manichanh et al. | Reduced diversity of faecal microbiota in Crohn’s disease revealed by a metagenomic approach | |
| CN104946739B (en) | EGFR genetic mutation detection kit and its application | |
| CN105331726A (en) | Method for sequencing human mitochondria genetic set based on sanger | |
| CN107557478A (en) | A kind of method and its application that amplification the Changjiang river fish chondriogen total order is combined based on degenerate primer | |
| CN105734679A (en) | Preparation method of nucleic acid target sequence capture sequencing library | |
| CN108570504A (en) | A kind of MGMT promoter methylations detection primer and its detection method | |
| CN102776286A (en) | Primer, probe and assay kit for detecting v-ros avian UR2 sarcoma viral oncogene homolog 1 (ROS1) gene fusion mutation | |
| Mochizuki et al. | Restriction fragment length polymorphism analysis of ribosomal DNA intergenic regions is useful for differentiating strains of Trichophyton mentagrophytes | |
| CN104531713A (en) | Multiple PCR primers and method for constructing human TCBR library based on high-throughput sequencing | |
| CN107557461A (en) | A kind of mass spectrographic detection method of nucleic acid early sieved for liver cancer susceptibility | |
| Peng et al. | Sequence variations of mitochondrial DNA D-loop region are associated with familial nasopharyngeal carcinoma | |
| CN106701903A (en) | Reagent kit for detecting mitochondrial heteroplasmy and detection method | |
| Lee et al. | Authentication of medicinal plants by SNP-based multiplex PCR | |
| Grenouillet et al. | Multiple-locus variable-number tandem-repeat analysis for rapid typing of Candida glabrata | |
| CN107177676A (en) | Long-chain non-coding RNA NONHSAT113026 is used for the purposes of Diagnosis of Renal Cell Carcinoma molecular marker | |
| CN103571958A (en) | Primer and kit for detecting methylation of MGMT (O<6>-methylguanine-DNA methyltransferase) genes and use method of primer | |
| CN106754903A (en) | The primer pair and method of a kind of whole genome amplification for human mitochondrial | |
| CN104894270B (en) | Primer pair used for identifying peucedanum decursivum and application thereof | |
| CN105177149B (en) | Application of the miRNA in liver cirrhosis diagnosis and treatment | |
| CN102925580A (en) | Quick detection method for Notch1 gene mutation | |
| CN104531698A (en) | Multi-PCR (Polymerase Chain Reaction) primer and method for constructing mouse TCRB (T-Cell Receptor Beta) library based on high-throughput sequencing | |
| CN104894121A (en) | Primer pair used for identifying peucedanum praeruptorum and application thereof | |
| US11021756B2 (en) | MiRNA markers for the diagnosis of osteosarcoma | |
| CN109797235A (en) | With the molecular labeling R112823 of resistance gene of rice blast Pi1 close linkage |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20160217 Assignee: Shanghai Puchuang Longke Finance Leasing Co.,Ltd. Assignor: SHANGHAI PERSONAL BIOTECHNOLOGY Co.,Ltd. Contract record no.: X2019310000027 Denomination of invention: Method for sequencing human mitochondria genetic set based on sanger Granted publication date: 20190212 License type: Exclusive License Record date: 20191225 |
|
| EE01 | Entry into force of recordation of patent licensing contract | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Method for sequencing human mitochondria genetic set based on sanger Effective date of registration: 20191227 Granted publication date: 20190212 Pledgee: Shanghai Puchuang Longke Finance Leasing Co.,Ltd. Pledgor: SHANGHAI PERSONAL BIOTECHNOLOGY Co.,Ltd. Registration number: Y2019310000041 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20201228 Granted publication date: 20190212 Pledgee: Shanghai Puchuang Longke Finance Leasing Co.,Ltd. Pledgor: SHANGHAI PERSONAL BIOTECHNOLOGY Co.,Ltd. Registration number: Y2019310000041 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| EC01 | Cancellation of recordation of patent licensing contract |
Assignee: Shanghai Puchuang Longke Finance Leasing Co.,Ltd. Assignor: SHANGHAI PERSONAL BIOTECHNOLOGY Co.,Ltd. Contract record no.: X2019310000027 Date of cancellation: 20210108 |
|
| EC01 | Cancellation of recordation of patent licensing contract | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A Method for Sequencing the Human Mitochondrial Genome Based on Sanger Effective date of registration: 20231016 Granted publication date: 20190212 Pledgee: The Bank of Shanghai branch Caohejing Limited by Share Ltd. Pledgor: SHANGHAI PERSONAL BIOTECHNOLOGY Co.,Ltd. Registration number: Y2023980061192 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right |